Resolution of ultrafast excited state kinetics of bilirubin in chloroform and bound to human serum albumin

The ZE-isomerisation of bilirubin upon excitation with visible light is a fundamental step in phototherapy of newborns with neonatal jaundice.

Here we report results of an ultrafast optical spectroscopy study of bilirubin in CHCl3 as well as bound to human serum albumin.

The data show that the initially excited singlet state has sub-ps decay times with major amplitude.

Transient absorption measurements reveal that the ultrafast decay of the emission is accompanied by the formation of a transient intermediate which decays on the 15–20 ps timescale.

The initial photoprocesses are thus considerably faster than the previously reported fastest lifetimes for bilirubin and this is, to our knowledge, the first time that the earliest processes in excited bilirubin have been resolved.

Bilirubin-IXα (BR, see Fig. 1 for the structure) is a yellow-orange, neurotoxic substance formed in mammals by the catabolism of haem.

It is an open-chain tetrapyrrole consisting of two nearly identical dipyrrinone chromophores with propionic acid side-chains located at C-8 and C-.121

The publication of the crystallographic structure in 1976 confirmed previous predictions that 4Z,15Z-BR adopts a three-dimensional ridge-tile shape stabilised by a network of six intramolecular hydrogen bonds between the carboxyl groups and the lactam and pyrrole moieties of the opposite dipyrrinone unit.2

This folded conformation is retained in chloroform,3 and even to a large extent in strongly polar solvents like DMSO and aqueous buffer.4,5

The persistence of hydrogen bonding, internally neutralising all the polar groups, is most likely the reason why BR is essentially insoluble in water at physiological pH and therefore intrinsically unexcretable.

In the normal clearance pathway, BR is transported through the bloodstream to the liver in the form of an association complex with serum albumin and excretion is facilitated through hepatic conjugation to glucuronic acid by bilirubin uridine-5′-diphosphate glucuronosyltransferase.

However, the levels of this enzyme are initially low in newborn infants and unconjugated BR accumulates in the blood.

This often leads to a characteristic yellowing of the skin and sclera of the eye, a condition known as neonatal jaundice.

Since the late 1960s phototherapy with white or blue light has emerged as an effective and widely used method to lower abnormally high BR levels in jaundiced infants.

Consequently, the determination of the principal mechanism(s) responsible for its success has been the focus of numerous investigations.6,7

It is currently believed that phototherapy enhances the excretion of unconjugated BR in bile and urine primarily through the formation of more polar configurational and structural photoisomers of BR.

Some photooxidation and destruction of the chromophore also occurs, but these processes are slow and do not make a major contribution.

Configurational photoisomerisation around one of the two exocyclic double bonds to give the 4E,15Z or 4Z,15E isomers is the most efficient photochemical reaction of BR in organic solution.

However, these methanol-soluble E-isomers are relatively unstable because one set of hydrogen bonds (Fig. 1) is broken upon ZE isomerisation.

Reversion to the 4Z,15Z configuration occurs thermally or photochemically and during irradiation a photoequilibrium is rapidly reached because these three isomeric forms have extensively overlapping absorption spectra.8

Upon prolonged photolysis, the formation of lumirubin, a more stable (irreversibly cyclised) structural isomer of BR, also becomes a significant process.9

Interestingly, both types of photoisomerisation process are enhanced when BR is bound to the high affinity site of human serum albumin (HSA).

Moreover, the ZE isomerisation occurs regioselectively and 4Z,15E-BR is formed almost exclusively with blue-light excitation.9,10

A complicating factor in the study of the photochemistry of BR is the unusual wavelength dependence of the reactions.11

The quantum yield of the 4Z,15E isomer of BR bound to HSA decreases from 0.11 at 457.9 nm to 0.054 at 514.5 nm.12

Conversely, the quantum yield of lumirubin formation increases13 in an approximately inverse fashion to a maximum of 0.002 at 514.5 nm.10

In addition, the isomeric ratio 4E,15Z: 4Z,15E of HSA-bound BR increases ten-fold over the same wavelength range.10

Of particular significance is the observation of a modest decline in the quantum yield of 4Z,15E-BR with increasing wavelength in ammoniacal methanol solution, suggesting the complex behaviour of BR is, at least partly, an inherent property of the molecule.14

The origin of these effects has been attributed to exciton coupling of the excited singlet states of the two dipyrrinone chromophores and to the extent of localisation of the excitation energy on the lower-lying C-15 bridged dipyrrinone.15,16

A dipole–dipole interaction energy (ΔEij) of ca. 1000 cm−1 has been calculated from the structure, assuming point dipoles and two identical chromophores,17 in fair agreement with the splitting energies (2ΔEij) used to simulate the absorption and circular dichroism spectra.17,18

Photophysical measurements of excited BR in solution at room temperature indicate that radiationless decay to the ground state is the dominant deactivation pathway, since the quantum yields of fluorescence (ϕf < 0.0002),15 intersystem crossing to the triplet state (ϕisc ≤ 0.01),19 and photoisomerisation (ϕZE + ϕEZ ≤ 0.035)14 are all very small.

The fluorescence yield of BR is, however, enhanced on binding to HSA (ϕf = 0.003) and there is a further dramatic increase (ϕf = 0.92) upon lowering the temperature to 77 K as a result of the increased environmental rigidity.15

It is therefore surprising that the lifetime of the lowest excited singlet state of BR determined by picosecond transient absorption is practically the same in CHCl3 (17 ± 3 ps) and bound to HSA (19 ± 3 ps) at room temperature,20 although a somewhat longer fluorescence lifetime was reported for HSA-bound BR using photon-counting methods.21

Nevertheless, the assignment of the transient absorption spectra to the singlet state appears to be supported by the 14 ± 2 ps fluorescence lifetime measured in CHCl3,21 and the 18 ± 3 ps decay time for HSA-bound BR obtained using a streak camera.15

Two important conclusions that were drawn from these and other measurements are (1) that the excited singlet state decays rapidly, with near unity efficiency, to a very short-lived twisted intermediate state from which decay back to the ground state configuration is much more favourable than conversion to the photoisomer and (2) the radiative rate of BR must increase by more than an order of magnitude on binding to HSA.15

To determine if these conclusions are valid, we have reinvestigated the early photoprocesses of BR in chloroform solution and bound to HSA using femtosecond transient absorption and fluorescence techniques.

Fluorescence decays were recorded with magic-angle polarisation at room temperature using the fluorescence upconversion technique and a spectrometer that has been previously described.22,23

Bilirubin (for biochemistry, Merck) and HSA (essentially fatty acid and globulin free, Fluka) were used as received and stock solutions were prepared fresh in dim light conditions.

The solutions were held in a rotating cell with a 0.5 mm path length and were changed regularly, because prolonged irradiation led to broadening in the absorption spectrum, photodegradation, and formation of biliverdin (in CHCl3).24

In addition, the energy of the excitation pulses were intentionally kept low (∼0.1 nJ) and the initial and final scans were checked to ensure that the kinetics had remained constant.

The normalised steady state absorption and fluorescence spectra of BR in CHCl3 and bound to HSA in a 1∶1 stoichiometry are shown in Fig. 2.

The absorption spectrum of HSA-bound BR is noticeably broader than in CHCl3, but the maximum is shifted only 1–2 nm to the red.

Consequently, an excitation wavelength of 454 nm, corresponding to the absorption maximum of BR in CHCl3, was chosen for the measurements.

There is a larger red shift of ca. 10 nm in the fluorescence maximum of HSA-bound BR (520 nm).

The fluorescence decay kinetics recorded at 525 nm with variable time steps (from 10 fs up to 1 ps), to enable improved coverage of the different time ranges, are presented in Fig. 3.

The decay of the BR fluorescence is clearly very fast and it is not single-exponential.

The intensity of the emission from HSA-bound BR falls to half the maximum value in approximately one picosecond and it decays considerably faster in CHCl3.

No difference in the fluorescence decay could be detected using a sample of chloroform treated with KOH, compared to the untreated solvent.

Lifetimes obtained by multiexponential least squares fitting, using a response function of 150–170 fs, are given in Table 1.

Four or five reasonably well-separated lifetime components were required to obtain good agreement between the fitted and measured data.

The lifetimes and amplitudes of the major components (those with >10% amplitude) are believed to be accurate within 15%, as judged by comparing results from independent measurements, as well as the quality of fits after fixing one of the major lifetime components at different values.

The two sub-picosecond lifetime components account for the major part of the amplitude in both cases.

Evidently, the decay of the BR fluorescence is more complex and occurs on a substantially faster timescale than previously reported using the time-correlated single-photon counting technique.21

A short singlet state lifetime is in fact expected from the extremely low quantum yield of fluorescence in chloroform (ϕf < 0.0002).15

The oscillator strength determined by integrating the visible absorption spectrum of BR and using the manufacturer’s absorption coefficient of εmax = 60 200 M−1 cm−1, is ca. 1.1 and the calculated25 radiative lifetime is τ0 ≈ 1.4 ns.

Thus, the singlet state lifetime of BR is estimated to be <280 fs.

This calculation does not take into account that there are two dipyrrinone chromophores contributing to the absorption transition.

However, even a doubling of the estimated lifetime does not reconcile this sub-picosecond lifetime with those lifetimes reported earlier.15,20,21

The calculated value is, however, in reasonable agreement with the weighted average of the two shortest lifetimes reported here.

The notably slower overall decay observed for BR bound to HSA is consistent with the more than ten-times enhancement in the fluorescence quantum yield of HSA-bound BR (ϕf = 0.003), compared to chloroform solution (ϕf < 0.0002),15 and thus it is not necessary to invoke a dramatically different radiative rate in HSA.

From the inset of Fig. 3, showing the normalised fluorescence decay on a longer time scale, it is apparent that the (smaller amplitude) medium and longer lifetime components are the major contributors to the steady state fluorescence of BR bound to HSA.

The situation is reversed in CHCl3, where the major part of the area under the decay curve is located within the first few picoseconds after excitation.

The slowing of the decay kinetics upon binding to HSA is attributed to steric hindrance, but there can be no major barrier to twisting, as the fluorescence quantum yield remains low and photoisomerisation is more efficient than in organic solvents.

Further evidence that the initial excited state processes of bilirubin occur on an ultrafast timescale is provided by femtosecond transient absorption experiments, which were performed using a 5 kHz Ti∶sapphire regenerative amplifier and optical parametric amplifier (Spectra Physics).23

BR in CHCl3 was circulated continuously through a 1 mm pathlength flow cell, at a flow rate sufficient to ensure a stable signal intensity, by means of a peristaltic pump.

The polarisation of the pump beam was set at the magic angle with respect to the probe beam and the response function was ca. 200 fs.

Fig. 4 shows time-resolved absorption difference spectra of BR in CHCl3 at different times ranging from ca. 50 fs to 25 ps after 454 nm excitation.

Immediately after excitation a negative signal (i.e. a decrease in absorption) appears at wavelengths shorter than 490 nm (marked (a) in Fig. 4).

Since the ground state of BR absorbs in this region (see Fig. 2), this signal can be assigned to ground state bleaching.20

A second negative absorption signal is also apparent between ca. 525 and 625 nm at ∼50 and 250 fs after excitation and this signal (marked (b) in Fig. 4) is attributed to stimulated emission.

Although this signal occurs in the same region as the steady state emission of BR (Fig. 2), the negative signal at 50 fs has a maximum at a longer wavelength and, moreover, it moves further to the red by 250 fs.

Overlapping the stimulated emission is an excited state absorption, which is apparent as a positive signal between ca. 490 and 520 nm in the ∼50 fs spectrum.

By 450 fs the positive absorption signal (marked (c) in Fig. 4) extends beyond 650 nm and, concommitant with the disappearance of the stimulated emission signal, continues to rise reaching a maximum by 2 ps, before decaying on a 15–20 ps timescale.

The time-resolved spectra that we measured at longer delay times (2 ps and longer) are in agreement with the picosecond absorption spectra of Greene et al.,20 showing both negative absorption (bleach) at wavelengths shorter than ca. 490 nm and positive absorption at longer wavelengths.

However, the stimulated emission signal was not observed in this earlier pioneering work and the positive transient absorption signal was assigned to the singlet state, rather than to formation of a transient intermediate.

Thus, it is not the excited singlet state of BR that has the same lifetime bound to HSA and dissolved in chloroform, but the twisted intermediate state.

A more detailed study of the time-resolved absorption of BR both in CHCl3 and complexed to HSA in buffer is in progress.

In conclusion, ultrafast time resolved fluorescence measurements have been performed on bilirubin dissolved in chloroform and bound to human serum albumin for the first time.

The decays are complex and dominated by sub-picosecond processes, but additional significantly slower components were observed for HSA-bound BR, accounting for the larger quantum yield of fluorescence.

The ultrafast disappearance of the emission was also seen in pump-probe absorption measurements of BR in CHCl3, as was the build up of a “dark state”, presumably a twisted intermediate, which decays on the 15–20 ps timescale.